Inactivated pepsin fragments for modulating immune system activity against human malignant tumor cells

ABSTRACT

Isolated anti-cancer peptides are disclosed which are characterized by the amino acid sequences TLTSGGGAIALPPSMAAPPLGPVAPLTGAIHAPTXG; TLSTATGGAIPPVAAMPPGLVAPTHGPAIHP; CCATSGPCGAVMILTPHLTA; MTLTTGSGAIAPAMPPGLPPHTGAIHAPM; and NXVPVSVEGYXQITLDSITX and a significant in vitro binding affinity for gp96. The peptides exhibit anti-tumor, anti-cancer activity in vivo. Also disclosed is an isolated antiviral peptide is characterized by the amino acid sequence GDEPLENYLDTEYF and a significant in vitro binding affinity for HIV-1 gp 120 and gp 41, and human CD4 cells. The peptide exhibits anti-retroviral activity in vivo, particularly anti-HIV-1 activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This utility patent application is a continuation-in-part applicationof, and claims priority from, pending U.S. Utility application Ser. No.11/177,427 filed on Jul. 11, 2005 and entitled “Irreversibly-inactivatedPepsinogen Fragment and Pharmaceutical Compositions Comprising the Samefor Detecting, Preventing, and Treating HIV”, the disclosure of which isincorporated herein in its entirety by reference, which in turn claimspriority from Provisional Application Nos. 60/626,882 and 60/635,938filed on, respectively, Nov. 12, 2004 and Dec. 15, 2004.

FIELD OF THE INVENTION

The present invention relates to compositions and methods comprising aninactivated pepsin fragment (“IPF”) for modulating immune systemactivity. More specifically, embodiments of the present invention aredirected to compositions and methods to elicit specific immunity torecognize peptides associated with tumors and malignancies. The presentinvention also relates to compositions and methods comprising IPF fortreating infections such as infection by the human immunodeficiencyvirus.

BACKGROUND OF THE INVENTION

Pepsin is a proteolytic enzyme produced in the mucosal lining of thestomach and acts to degrades protein, together with chymotrypsin andtrypsin. During digestion, these enzymes, each of which is effective insevering links between particular types of amino acids (e.g.phenylalanine, tryptophan and tyrosine), collaborate to break downdietary proteins to their components, i.e., peptides and amino acids.

Current studies indicate that immune protection against cancer requiresthe generation of a potent cellular immune response against a uniquetumor antigen expressed by a malignant cell. Thus, successful immuneprotection would first require identifying a unique antigen in the tumorcells (tumor specific antigen) and then inducing a potent T-cellresponse targeted to the tumor antigen. These tumor-associated antigens,however, would still be recognized by immune cells as ‘self’ molecules,and so no true activation of the immune system would occur. Thus, twoobstacles in targeting these tumor-associated molecules as a vaccineinclude the unresponsiveness of the immune system to ‘self’ molecules,which restricts its ability to generate potent cellular immuneresponses, and preventing the generated immune response from beingdirected to normal cells that express the target antigen.

Proteins that show promise in overcoming these problems include heatshock proteins (HSPs). HSPs include a collection of ubiquitouslyexpressed cytoprotective proteins, which are expressed by cells underconditions of cell stress, such as increased temperature, viralinfection and oxidative stress. Certain HSPs have been shown to haveimmunomodulatory effects, such as the induction of cytokines and thepromotion of cell activation and maturation (see, Pockley A G, Lancet363 (9382) 469-476 (2003)).

For example, Zheng et al. (2001) report that cell surface targeting ofHSP gp96 induces dendritic cell maturation and antitumor immunity asdemonstrated by the expression of immune factors such as interleukinsand certain cell surface antigens (e.g., CD40, CD80 and MHC class IIantigens). It has been known for some time that heat shock proteins bindpeptide and that heat shock proteins purified from cells chaperone alarge number of peptides derived from the cells from which they areisolated. This is the so-called ‘antigenic repertoire’ of that cell.Studies have demonstrated that immunizing mice with HSP70, HSP90 andgp96 isolated from murine tumor cells induces anti-tumor immunity andtumor-specific cytolytic T-cells. These studies also show that theimmunity results from tumor-derived peptides associated with the heatshock protein rather than from the heat shock proteins themselves. Morerecently, studies reported the use of calreticulin, HSP110 and grp170 inheat shock protein-based cancer immunotherapy. Specific immunogenicityof tumor-derived heat shock protein preparations have been studied inrelation to fibro sarcomas, lung carcinoma, prostate cancer, spinal cellcarcinoma and melanomas in mice and rats of different haplotypes. Thesestudies included chemically-induced tumors, UV-induced tumors, andspontaneous tumors. Heat shock proteins show promise in thatpreparations isolated from a given cell may be associated with a rangeof peptides, including self and antigenic peptides and in thatHSP-peptide complexes are highly immunogenic.

Certain heat shock proteins demonstrate “superantigen” activity. Theyare capable of activating large numbers of T-lymphocytes in a majorhistocompatibility complex-restricted manner. This polyclonal activationof certain T-cell subsets may be responsible for some of theimmunomodulatory effects. These components have been reported tostimulate immune responses to certain neoplasms and may be involved inthe pathogenesis of certain autoimmune diseases.

Gp96 is a HSP of particular interest. Gp96 is a 96 kDa glycoproteinlocalized to the endoplasmic reticulum, which can also be found at thecell surface. Gp96 is released into the extra cellular space duringnecrotic cell death and activates dendrite cells and macrophages byrealizing inflammatory cytokines and inducing dendrites cells to mature.Gp96 has the ability to transfer antigenic peptides for their MHC-classI-restricted presentation and allows gp96 to function as an efficientmessaging system alerting the immune system of an infection. Thisincludes the receptor-mediated uptake of gp96 by dendrite cells. Thereceptor is CD91, which is known as the α2 macroglobulin (α2M) receptorexpressed on phagocytes. The presentation of gp96-associated peptide byantigen-presenting cells (“APC's”) is induced by α2 macroglobulin. Gp96is bound by CD91 on dendrite cells and internalized. Gp96 induces theexpression of co-stimulatory molecules and the release of interleukin 12(IL-12) and tumor necrotic factor α (“TNFα”) by the APC.

Certain infections, such as by the human immunodeficiency virus, havealso presented challenges in targeting the disease-causing organism andneutralizing it. Typically, infection with the human immunodeficiencyvirus, HIV-1, eventually causes acquired immunodeficiency syndrome(AIDS) and an associated syndrome, AIDS-related complex (ARC).Neutralizing this virus has proved difficult, largely because itsstructure obstructs immune system access to viral epitopes and itsgenetic material is highly variable. Accordingly, researchers have beenseeking prophylactic and therapeutic methods for preventing orcontrolling HIV which are not dependent upon antibody-mediated immunity.

The HIV retrovirus replicates in certain immune system cells,specifically the CD4+ subset of T-lymphocytes (pre-Th cells arising inthe thymus). In the usual course of a cell-mediated immune response toan intracellular pathogen such as a virus, dendritic cells(antigen-presenting cells) carrying antigen fragments and secretedcytokines activate these CD4+ T-cells. Activated cells, called T-helperor Th cells, in turn secrete their own cytokines and stimulatemacrophages. CD4+ Th cells also propagate cellular immune response bybinding chemotactic cytokines (chemokines, CCs) to their CC surfacereceptors. It is by this route that HIV-1 infection of these cells isenabled because, in addition to binding chemokines, these CC receptorsact together with the CD4+ surface glycoprotein as co-receptors forHIV-1 and mediate entry of the virus into the CD4+ Th cell. There, thevirus usurps the native genetic material for viral replication whiledestroying cell functions essential for building immunity; theincreasing destruction of these cells appears to be responsible for theeventual collapse of the cell-mediated immune system often seen interminal AIDS patients.

It has been recognized that denying entry into CD4+ cells to the HIV-1virus could at least slow the progress of the infection and alleviate,if not cure, the disease and/or its symptoms. The complex mechanism bywhich the virus crosses the cell membrane has been widely investigated.Broadly, the entry of human immunodeficiency virus into, for example,CD4+ Th1 cells (T-helper type 1 cells), is dependent upon a sequentialinteraction of the gp120/gp41 subunits of the viral envelopeglycoprotein gp160 with the CD4+ Th1 cell surface glycoprotein and thecell surface receptor CCR5. On binding of gp120 with its cell surfacebinding sites, a conformational change in the latent gp41 subunitthrough an intermediate state to an active state is initiated, inducingfusion of the viral and cellular membranes and transport of the virusinto the cell (Weissenhom et al., Nature, 387:426-30 (1997)).

Accordingly, numerous binding experiments have been conducted in aneffort to find antiviral ligands that will effectively compete with theHIV-1 for CD4+ gp and/or CCR5 binding sites, or that will preferentiallyblock gp120 and/or gp41 binding domains. In one example, a reportedstructure (X-ray crystallography) comprising an HIV-1 gp120 corecomplexed with a two-domain fragment of human CD4 and an antigen-bindingfragment of a neutralizing antibody that blocks chemokine-receptorbinding, is said to reveal a CD4-gp120 interface, a conserved bindingsite for the chemokine receptor, evidence for a conformational change onCD4 binding, the nature of a CD4-induced antibody epitope, and specificmechanisms for viral immune evasion, “which should guide efforts tointervene” (Nature 393 (6686):632-1, 1998). Also, it has been shown thatinhibition of the change in structure of gp41 from its intermediate toactive state with peptides used as competitors for critical cellreceptors may reduce viral load, and that while gp120 masks epitopes onthe gp41 subunit in its latent state, gp41 may be vulnerable toneutralizing antibodies in its transient or intermediate state(Molecular Membrane Biology 16:3-9, 1999).

BRIEF DESCRIPTION OF THE DRAWINGS

Some aspects of the present invention are generally shown by way ofreference to the accompanying drawings in which:

FIG. 1 illustrates the porcine pepsinogen sequence, and major and minorsequences of this pepsinogen.

FIG. 2 is a photograph of an electrophoresis gel showing an inactivatedpepsinogen fragment (“IPF”) in the 45.0 kDa band. SDS-PAGE was used fordetermination of molecular weight of the components of IPF. Column 1included weight standards shown in the box to the left. The next twocolumns show #5, bovine albumin, at 1:300 dilution and 1:500 dilution.The remaining columns show samples 16-21 at various concentrations.Sample 16 was obtained from porcine pepsin Sigma p7000 1:10000 purifiedand methylated. Samples 17 and 21 were prepared as with Sample 16.Sample 18 was porcine pepsin Sigma p7000 1:10000, diluted in buffer atpH 3.2, so that pepsin is an active protease. Sample 19 was obtainedfrom porcine pepsin Sigma p7000 1:10000 and purified, but unmethylated.Sample 20 was prepared as with Sample 19. The next columns, in order,show methylated IPF sample #16 at a 1:1 dilution, methylated IPF sample#17 at a 1:1 dilution, untreated pure (active) pepsin sample #18 at a1:10 dilution, unmethylated IPF sample #19 at a 1:10 dilution,unmethylated IPF sample #20 at a 1:10 dilution, and methylated IPFsample #21 at a 1:10 dilution. IPF samples #16, 17, and 21 include as amajor component a protein/peptide migrating with an apparent molecularweight of 45 (k. These samples show highly specific binding between IPFand gp41, gp120, and CD4 at different concentrations.

FIG. 3 is a Biacore graph showing a HPLC (High Performance LiquidChromatography) chromatogram of an isolated IPF in accordance with onceaspect of the present invention.

FIGS. 4, 5, 6, and 7 illustrate exemplary binding of four samples of IPFwith gp41, gp120, human CD4, and human serum at 3 different dilutions;

FIG. 8 is a photograph of an electrophoresis agarose gel showing boundIPF and gp41.

FIG. 9 is a graph showing binding of an IPF with heat shock protein gp96at dilutions of IPF 1/2500, IPF 1/500 and IPF 1/100, according to oneembodiment of the invention.

FIG. 10 shows amino acid normalization for the 45 kDa IPF fragmentaccording to one embodiment of the invention.

SUMMARY OF THE DISCLOSURE

The present invention includes methods and compositions comprisingmodified cellular shock protein gp96 which preferably includes anirreversible pepsin fraction IPF and more preferably includes an IPFgp96complex. In another preferred embodiment, a complex of IPFgp96 may becombined with at least one other polynucleotide like a molecularadjuvant, such as IL-2, to increase cellular immune response.

Preliminary clinical trials have demonstrated the induction of cancerspecific CD8+ T-cells responses in 6/12 patients immunized withgp96-peptide prepared from their own tumor. The capacity oftumor-derived heat shock proteins to induce specific and protectiveimmunity might have profound effects on the treatment and management ofpatients with malignant disease. For example, studies have shown thatinduction of immunity to methylcholanthrane-induced fibro sarcoma by theadministration of gp96 isolated from the tumor displays consistent doserestriction: two intradermal administrations of <1 μg gp96 was found tobe ineffective; two doses of 1 μg was found to induce immunity andprovide optimal protection against tumor growth; and two doses of 10 μgwas found not to protect.

The lack of protection at high doses of tumor-derived gp96 is an active,antigen specific down-regulation of tumor-specific immunity that can beadoptively transferred by CD4 T-cells purified from animal treated withhigh doses of tumor derived gp96. These findings are exciting as theysuggest that immunization with heat shock proteins that are chaperoningclinically relevant peptides might be an effective strategy for downregulating several diseases including autoimmunity.

The addition of IL-2 to activate normal human lymphocytes directlypromotes several other cellular functions as well as proliferation. IL-2stimulated T-cells exhibit enhanced cytotoxicity and produce lymphokinssuch as INF-γ TNF-β and TGF-β; B-cells growth factors such as IL-4 andIL-6 and GM-CSF. IL-2 also induces lymphokine-activated killer (LAK)activity, which is predominantly due to NK cells.

The present invention also encompasses a cancer preventive ortherapeutic vaccine comprising a IPFgp96 complex, and more specificallycomplexes of IPF-1 gp96; IPF-2gp96; IPF-3gp96, IPF-4gp96, IPF-5gp96,singly or in combinations thereof, which may be mixed with one or morepolynucleotides encoding a molecular adjuvant. Any molecular adjuvantthat increases cellular immune response may be used like cytokine IL-2.In the preferred embodiments, the compositions and methods of thepresent invention comprise binding between IPF fragments and receptorsof gp96, such as for example, CD91, in vivo. Administration may be viaan intramuscular injection. The cancer to be treated may be primary ormetastatic and the patients to be treated may have multiple differenttypes of cancer.

IPF has been shown to have an ability to modulate Th1 immunity, cytokinesecretion and γIFN. Placebo and controlled double blind assay using twogroups of rats show immunological changes following active therapy,which were sustained over time. These changes include: 1) increase inthe CD4+ CD45RO+ CD62 L population; 2) increase in the CD4+ CD45RA+ CD62L population; 3) appearance of a second CD4+ population having lower CD4intensity but no increase in SSC, implying a second CD4 cell population.Preliminary studies of this population in isolation reveals that thesecells are not memory or naïve cells; 4) a parallel increase in absoluteCD4 cell counts; 5) an increase in CD8+ CCR5+ population.

In addition, functional assays showed the following: 1) an increase inthe IFN-[y] containing CD3+ CD4+ cells post stimulation in vitro; 2) adecrease in the IL-4 containing CD3+ CD4+ cells post-stimulation; 3) asignificant increase in the IFN-γ containing CD3+ CD8+ cells over time.

Also, numerous binding experiments have been conducted in an effort tofind antiviral ligands that will effectively compete with the HIV-1 forCD4+ gp and/or CCR5 binding sites, or that will preferentially blockgp120 and/or gp41 binding domains. In one example, a reported structure(X-ray crystallography) comprising a HIV-1 gp120 core complexed with atwo-domain fragment of human CD4 and an antigen-binding fragment of aneutralizing antibody that blocks chemokine-receptor binding, is said toreveal a CD4-gp120 interface, a conserved binding site for the chemokinereceptor, evidence for a conformational change on CD4 binding, thenature of a CD4-induced antibody epitope, and specific mechanisms forviral immune evasion, “which should guide efforts to intervene” (Nature393 (6686):632-1, 1998). Also, it has been shown that inhibition of thechange in structure of gp41 from its intermediate to active state withpeptides used as competitors for critical cell receptors may reduceviral load, and that while gp120 masks epitopes on the gp41 subunit inits latent state, gp41 may be vulnerable to neutralizing antibodies inits transient or intermediate state (Molecular Membrane Biology 16:3-9,1999).

Other embodiments of the present invention are generally directed toproviding pharmaceutical compositions comprising IPF (IPF-1, IPF-2,IPF-3, IPF-4, IPF-5 and/or IPF-6) and methods for preventing, treating,and diagnosing HIV-1 infections and HIV-1 related conditions such asAIDS (Acquired immune Deficiency Syndrome) and ARC (AIDS RelatedComplex) with these compositions.

In an exemplary embodiment the present invention relates to a method ofmodulating immune system activity comprising administering to a patientan effective amount of a composition containing inactivated pepsinfragment (IPF).

The isolation, purification and characterization and a variety of otheruses, e.g., diagnosis and treatment of HIV infection and relateddiseases such as AIDS and ARC, of the inactivated pepsin fragment orfraction (IPF) as used herein is described in commonly owned U.S.Provisional Patent Application No. 60/644,054, filed Jan. 18, 2005,Zhabilov, the contents of which are incorporated herein by reference intheir entirety.

At least 8 isozymes of pepsinogen have been identified in gastricepithelial cells, and these have been categorized into twoimmunologically-separable types (pepsins A and C). The mature, activeenzymes are roughly 325 amino acids with a mass of approximately 35 kDa.Pepsins are synthesized as inactive pre-proenzymes, consisting of asignal peptide, activation peptide and active enzyme. The signal peptideis cleaved as the protein is inserted into endoplasmic reticulum and theresulting proenzyme—pepsinogen—is transported to the Golgi and condensedinto secretory granules. Pepsinogens are secreted in a form such thatthe activation peptide assumes a compact structure that occludes theactive site. On exposure to an acidic (pH <4) environment such as occursin the lumen of the stomach, the activation peptide unfolds, allowingthe active site to clip it off, yielding mature, catalytically activepepsin.

Structurally, the active site is located in a deep cleft within themolecule located between two homologous portions of the structure, theN-terminal lobe (residues 1-172) and the C-terminal lobe (residues173-327). Optimal activity of pepsins is at pH of 1.8 to 3.5, dependingon the isoform. They are reversibly inactivated at about pH 5 andirreversibly inactivated at about pH 6 to 7 or 7 to 8. See, Yuji O.Kamatari, Christopher M. Dobson, and Takashi Konno, “Structuraldissection of alkaline-denatured pepsin,” Protein Science (2003),12:717-724.

According to Entrez Protein, accession number NP_(—)999038, a sequenceof swine porcine pepsinogen is SEQ ID NO:1—

000 mkwllllslv vlseclvkvp lvrkkslrqn likngklkdf lkthkhnpas kyfpeaaali061 gdeplenyld teyfgtigig tpaqdftvif dtgssnlwvp svycsslacs dhnqfnpdds121 stfeatsqel sitygtgsmt gilgydtvqv ggisdtnqif glsetepgsf lyyapfdgil181 glaypsisas gatpvfdnlw dqglvsqdlf svylssndds gsvvllggid ssyytgslnw241 vpvsvegywq itldsitmdg etiacsggcq aivdtgtsll tgptsaiain iqsdigasen301 sdgemviscs sidslpdivf tingvqypls psayilqddd sctsgfegmd vptssgelwi361 lgdvfirqyy tvfdrannkv glapva

According to Kamatari et al., after the N-terminal 60 bases ofpepsinogen are cleaved off to produce pepsin, the N-terminal lobe ofpepsin protein includes residues 1-172, and the C-terminal lobe includesthe remaining residues 173-326. IPF-6 according to the invention differsfrom pepsin. It includes a major component having an apparent MW of 45kD when subjected to SDS-PAGE as shown in FIG. 2. It is unclear whetherthe 45 kD IPF-6 peptide is actually larger than pepsin, e.g. due todimerization or other bonding with itself or another peptide, or whetherthe 45 kD apparent Molecular weight is an artifact due to chemicalmodification of the protein such as methylation. The term “45 kD IPF-6”is used here to refer to the IPF-6 molecule obtained and assayed asdescribed here, whether its actual molecular weight is 45 kD or anotherfigure.

Surprisingly, preparations isolated from pepsin provide highlysensitive, specific, and therapeutic biological preparations. Thestarting material may be pure, active porcine pepsin A. An exemplarystarting material is Sigma porcine pepsin P 7000, which has aconcentration of 1:10000. Other starting material preparations areacceptable and may be effective according to the invention, if treatedand isolated according to the invention. For example, porcine pepsinwith other concentrations may be used e.g. 1:60000. The startingmaterial is preferably white to yellow, not brown, and may have amilk-like smell.

Other natural or recombinant sources of pepsin may be used, or othersimilar aspartic proteases, provided that the protease is inactive atalkaline, neutral, near-neutral, or mildly acidic pH, has a pI in thoseranges, and has a sequence with substantial homology to the IPF 45 kDfragment reported here. Substantial homology means at least about 50,60, 70, 80, 90, or 95% identity of amino acid residues in the relevantportions of the molecule, or structural homology. Pepsin sharesstructural homology with HIV and other aspartic proteases. Campos andSancho, “The active site of pepsin is formed in the intermediateconformation dominant at mildly acidic pH,” FEBS Letters Vol. 538:89-95(2003).

The invention provides several uses for the peptides. Examples ofpossible uses include a diagnostic assay and a therapeutic agent. IPFcauses a dramatic rise in cytokines (e.g. interleukin 9 and 10) andantibodies to p24 antigen in HIV patients.

The properties of the peptides identified as part of the IPF hereininclude MW, inactivity at neutral pH, as present in blood, and theirpartial sequence data. Also, the IPF fragments migrate as a single mainpeak in HPLC, such as shown in the chromatogram of FIG. 1 for IPF-6,with a particular retention time. Because pepsin is inactivated duringisolation of IPF, the preparation is stable and does not degradesignificantly over time.

The inactivated pepsin fragments (IPF) of the invention may be referredto as irreversibly-inactivated. This is due to its treatment at neutralpH. As noted above, inactivation occurs above about pH 5, and becomesirreversible above about pH 6, and proteolytic activity is lost by suchtreatment. Maximal activity of pepsin as an enzyme is between 2-4 pH.The inventive method increases the pH of diluted pepsin to above 5,above about 6, and desirably in the range of pH 6.6-6.8 beforeprecipitation and during use. Thus IPF formulation pepsin fragments areirreversibly inactivated.

The treatment, fragmentation, and isolation procedures inactivate pepsinand cut the pepsin chain into separate peptide fragments. IPF may beisolated from active pepsin. For example, Sigma porcine pepsin P7000 maybe used. This is a pepsin A from porcine gastric mucosa, is a powderwith 800-2,500 units/mg protein. The CAS number is 9001-75-6, and the ECnumber is 3.4.23.1. According to Sigma, it preferentially cleavesC-terminal to Phe, Leu and Glu. It does not cleave at Val, Ala or Gly.Optimum pH is 2-4. Stable at 60° C. Pepsin is irreversibly inactived atpH >6 and has a mol wt 35 kDa. See, Harlow, E., and Lane, D.,Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y. (1988),626-628; and Merck 13, 7225.

The IPF 45 kD peptide and IPF 15 kD peptide have amino acid sequenceshomologous to pepsin, meaning that at least about 5, 10, 15, or 20 ofthe amino acid residues in the peptide are identical to those of pepsin.

As used herein, the present invention may be directed to modulatingimmune system activity, which includes treating, decreasing, increasing,attenuating or modulating any condition that may benefit from anenhancement of immune system activity. Immune conditions can includeimmune diseases or disorders. Immune disorders may include Allergies,Auto-Immune, DiGeorge Syndrome, Familial Mediterranean Fever, ImmuneDeficiency, and Multiple Chemical Sensitivity.

Immune system disease or disorder may include at least one ofAgammaglobulinemia, Anaphylaxis, Antiphospholipid Syndrome, AtaxiaTelangiectasia, Autoimmune Diseases, Common Variable Immunodeficiency,DiGeorge Syndrome, Electrosensitivity, Familial Mediterranean Fever,Graft vs Host Disease, Granulomatous Disease, Chronic, HIV Infections,Hypersensitivity, Hypersensitivity, Immediate, IgA Deficiency, ImmuneComplex Diseases, Immune System Diseases, Immunologic DeficiencySyndromes, Lambert-Eaton Myasthenic Syndrome, Lambert-Eaton MyasthenicSyndrome, Latex Hypersensitivity, Lymphoproliferative Disorders,Multiple Chemical Sensitivity, Purpura, Schoenlein-Henoch, Samter'sSyndrome, Severe Combined Immunodeficiency, Sick Building Syndrome,Sjogren's Syndrome, and Wiskott-Aldrich Syndrome.

In one aspect of the invention, auto-immune disorder may compriseAddison's, Ankylosing Spondylitis, Antiphospholipid Syndrome, BarthSyndrome, Graves' Disease, Hemolytic Anemia, IgA Nephropathy, LupusErythematosus, Systemic, Microscopic Polyangiitis, Multiple Sclerosis,Myasthenia Gravis, Myositis, Osteoporosis, Pemphigus, Psoriasis,Rheumatoid Arthritis, Sarcoidosis, Scleroderma and Sjogren's Syndrome.Examples of allergies may include Asthma, Food, Hay Fever—Rhinitis,Hives, Latex and Sinusitis. In yet another embodiment, the patient mayhave AIDS or AIDS Related Complex, multiple sclerosis, hepatitis,herpes, rheumatoid arthritis, autoimmune diabetes, encephalomyelitis oranother autoimmune disease.

In another exemplary embodiment, the present invention may encompass acancer preventive or therapeutic vaccine.

In yet another exemplary embodiment, the IPF is administered with atleast one other polynucleotide, like a molecular adjuvant, for cancerpreventive or therapeutic vaccine. The cancer can be either primary ormetastatic and may include renal cell carcinoma (kidney cancer),melanoma, pancreatic cancer, non-Hodgkin's lymphoma, lung carcinoma,prostate cancer, spinal cell carcinoma, soft tissue sarcoma orfibrosarcoma.

Compositions comprising the IPF fractions disclosed herein may also beused to treat hepatitis, multiple sclerosis, lupus, and herpes simplex.Anecdotal observations, backed up by blood work, indicate a reduction inseverity of symptoms in patients with these diseases, and predictsefficacy in other viral and autoimmune diseases.

In an exemplary embodiment, IPF peptides, e.g. IPF1-6, show specificbinding and indicate usefulness as a) a diagnostic and b) a therapeuticfor HIV, and other diseases. The binding of this protein material withenvelope proteins in several infectious diseases as well as directbinding to CD4 cells indicates that IPF can stimulate an immunologicalreaction, for example by promoting the formation of superantigens whichincrease production of specific antibodies.

Diagnostic methods using IPF may be performed by combining IPF with testand control sera and conducting 2-D electrophoresis in 1% agarose gels,following the techniques set forth in Zhabilov et al. (US 2004/0018639,filed 3 Jun. 2003 by Zhabilov, Harry P. et al. and incorporated hereinby reference), with modifications apparent to a person of ordinaryskill. Therapeutic methods using IPF disclosed herein are performed byadministering IPF pharmaceutical compositions to a subject having adisease susceptible to treatment with IPF. The formulations, dosages,dosing regimen, and routes of administering may be those described inZhabilov et al. or other examples apparent to a person of ordinaryskill.

In another exemplary embodiment, the IPF composition may be in a varietyof forms, e.g., a pharmaceutical composition. In one aspect, thepharmaceutical composition may comprise the IPF disclosed herein and apharmaceutically effective carrier, e.g., buffered saline, water,aluminum hydroxide, or another suitable adjuvant.

In yet another exemplary embodiment, the composition may containpreservatives, vehicles, buffers, tonicity adjusters, chelating agents,antioxidants and or other material. Examples of preservatives includePhenylethyl alcohol USP, Sorbic Acid NF, Sodium Propionate, SodiumBenzoate NF, and Benzyl alcohol NF. Examples of Vehicles includePurified Water USP, Hydroxy Ethyl Cellulose NF, Polyethylene Glycol NF,Povidone USP, Hydroxypropyl Methylcellulose F4M USP, Dextran 70 USP,Poloxamer NF, Polyoxyl-40-Stearate USP and Aluminum Chloride. Examplesof buffers include Sodium Phosphate (mono, di and tribasic), SodiumCarbonate, Sodium Biphosphate, Sodium Bicarbonate USP, Citric AcidMonohydrate USP, Acetic Acid, Sodium Citrate USP, Phosphoric Acid,Glacial Acetic Acid USP, Sodium Hydroxide NF, Sodium Acetate USP,Potassium Citrate USP, Hydrochloric Acid NF, and Potassium Phosphates:(mono, di and tribasic). Examples of tonicity adjusters include SodiumChloride USP, Dextrose USP, Glycerin USP, Potassium Chloride USP andMannitol USP. Examples of chelating agents include Edetate Disodium USP,Edetate Monosodium, Edetic Acid NF, and Edetate Trisodium. Examples ofantioxidants include Sodium Metabisulfite NF, Sodium Bisulfite, SodiumThiosulfate USP, and Acetylcysteine USP. Other material may includePolysorbates (20-85) NF, Pluronic F168, Pluronic F127, and PolyethyleneGlycol 300, 400, 6000 NF.

The IPF compositions disclosed herein may be administered in a varietyof manners, e.g., orally, by inhalation, intradermally, intramuscularly,subcutaneously or intravenously. It may be in the form of an injectablesolution or formulation, tablet, liquid formulation, lyophilized oraerosolized receptors.

In one embodiment, the IPF compositions disclosed herein areadministered intramuscularly. Also, doses may be administered at leastdaily, weekly or monthly, for as long as treatment is required. Inexemplary embodiments, the IPF is administered intramuscularly once aweek for six week, twice weekly for eight weeks, or as sixteeninjections with two injections on consecutive days per week for eightweeks.

The IPF disclosed herein may be administered via the composition in avariety of doses, e.g., from about 1 to about 25 mg of per 1 ml of thecomposition. In an exemplary embodiment, the IPF is administered inabout 8 mg or about 4 mg per 2 ml of formulation. The IPF may beadministered in an amount of 57 μg per 1 kg of body weight of thepatient. In mice, e.g., IPF can be administered from about 0.1 to about0.5 mg/kg of body weight and in rabbits, about 1.24 mg/kg, twice weeklyfor eight weeks.

The IPF disclosed herein may have one or more of the following effects:suppressing tumor immunity or eliciting protective immunity againsttumor cells, chaperoning immune enhancing agents and peptides,activating dendrites and macrophages by modulating inflammatorycytokines and inducing maturation of dendrites, modulating release ofIL-12 and tumor necrosis factor α (TNFα), inducing anti-tumor activityand tumor-specific cytolytic T-cells and inducing cancer-specific CD8⁺T-cell response.

The IPF components disclosed herein may have one or more of thefollowing phenotypic effects: increasing the CD4+ CD45 RO+ CD62 Lpopulation, increasing in CD4+ CD45 RA+ CD62 L population, inducing asecond CD4+ population having lower CD4 intensity but no increase inSSC, inducing a parallel increase in absolute CD4 cell counts when thisphenomenon appears, and increasing the CD8+ CCR5+ population.

The IPF component may have one or more of the following functionaleffects over time: increasing the IFN-γ containing CD3+ CD4+ cells poststimulation in vitro, decreasing the IL-4 containing CD3+ CD4+ cellspost stimulation, and increasing the IFN-γ containing CD3+ CD8+ cellsover time.

DETAILED DESCRIPTION OF THE INVENTION

Pepsins (of which there are several isozymes) are the principalproteases in gastric secretions of adult mammals. They belong to thefamily of aspartic proteases and are synthesized and secreted by cellsin the gastric mucosa as inactive enzyme-precursors consisting of asignal peptide, an activation peptide and an occluded active enzyme. Enroute to the lumen of the stomach for protein digestion, the signalpeptide is cleaved to yield the inactive proenzyme pepsinogen, which, onexposure to a low gastric pH (<4), cleaves in turn to yield mature,catalytically active pepsin.

Porcine pepsin was one of the first enzymes to be studied, and isperhaps the best-understood aspartic protease. It has 327 amino acid(aa) residues, and a molecular mass of 34 kDa (PNAS (U.S.) 70:3437-391973). Proteolytic activity of pepsin is at its highest at a pH of about1.8 to 3.5; it is inactivated at a pH of about 5 and irreversiblyinactivated (denatured) at a pH of about 6-7. Owing to their importance,amino acid residues associated with the substrate binding (active) sitehave been a research focal point. However, it is apparently still notclear how much functional activity, if any, is influenced by theremainder of the peptide.

The family of aspartic proteases (aspartases) is characterized byaspartic acid residues at their active (catalytic) sites. Human pepsin,for example, has two active site aspartate residues (coded “D” or“Asp”). This family also includes the HIV protease (and its numerousvariants), comprising two identical chains each having a singleactive-site aspartate residue. Essential for maturation of the newlysynthesized virus, which is expressed as a polyprotein, this proteasehas become a popular target for researchers attempting to block HIVreplication.

Certain embodiments of the present invention are generally directed toproviding isolated peptides characterized by, respectively, the aminoacid sequences TLTSGGGAIALPPSMAAPPLGPVAPLTGAIHAPTXG (SEQ ID: NO 1), afragment of approximately 45 kDa; TLSTATGGAIPPVAAMPPGLVAPTHGPAIHP (SEQID: NO 2); NXVPVSVEGYXQITLDSITX (SEQ ID: NO. 3), a fragment ofapproximately 13.5 kDa; MTLTTGSGAIAPAMPPGLPPHTGAIHAPM (SEQ ID: NO. 4);and CCATSGPCGAVMILTPHLTA (SEQ. ID: NO. 5), and a significant in vitrobinding affinity for gp96. The peptides, referred to herein as,respectively, IPF-1, IPF-2, IPF-3, IPF-4 and IPF-5 (InactivatedPepsinogen Fragments-1-5), were isolated from porcine pepsinogen,purified, and irreversibly inactivated for use in cancer therapeuticprocedures.

The present invention also encompasses a cancer preventive ortherapeutic vaccine comprising IPFgp96, and more specifically IPF-1gp96;IPF-2gp96; IPF-3gp96, IPF-4gp96 and IPF-5gp96 or combinations thereof,which may be mixed with one or more polynucleotides encoding a molecularadjuvant. Any molecular adjuvant that increases cellular immune responsemay be used like cytokine IL-2. Administration may be via anintramuscular injection. The cancer to be treated may be primary ormetastatic and the patients to be treated may have multiple differenttypes of cancer.

The heat shock protein, e.g., gp96, may be prepared according tosuitable methods known in the art, such as according to the methods setforth in Chandawarkar, et al. (Int'l Immunology, Vol. 16, No. 4, 615-624(2004)), incorporated in its entirety by this reference. Complexescomprising HSP and IPF may be prepared according to suitable methodsknown in the art, such as disclosed in application No.PCT/US2006/038045, also incorporated in its entirety by this reference.

FIG. 9 is a graph showing binding of an IPF with heat shock protein gp96at dilutions of IPF 1/2500, IPF 1/500 and IPF 1/100, according to oneembodiment of the invention. A number of approaches may be used todetect complexing between IPF and gp96. By way of example, one suchapproach may be to obtain specific antibodies against IPF and gp96 andto build a sandwich immunoassay by suitable methods known in the art todetect the presence of these protein complexes. To detect binding ofgp96 to IPF, the antigen may be coated on a plate to capture proteinsfrom the sample and then report the binding with a specific detectionantibody. The secondary (or detection) antibody may be directly labeledwith MSD SULFO-TAG NHS ester or a SULFO-TAG-labeled anti-species, e.g.,anti-mouse if the detection antibody is raised in mouse and thedetection antibody is raised in a different species than the capture orprimary antibody. Antibodies specific to IPF-gp96 complexes may also beused.

The cancer preventive vaccine may comprise a clear liquid opalescentsuspension of spontaneous precipitate IPF (e.g., IPF-1, IPF-2, IPF-3,IPF-4 and/or IPF-5) and gp96 molecules with IL2 as adjuvant and maycomprise complexes of IPF-1, IPF-2, IPF-3, IPF-4 and IPF-5 and gp96.Activity is preferably 1:0.6 measured by the ability of IPF to bind withgp96 (e.g., three molecules of IPF bound two molecules of gp96). Thecancer preventive vaccine may be injected intramuscularly one injectionper week for six weeks. The immune response probably gives evidence oftwo actions: 1) cytotoxic effect against tumor cells (cytotoxic Tlymphocytes (CTLs) are effectors of CD8+ that can mediate the lysis oftarget cells bearing antigenic peptides associated with a MHC molecule.Other cytotoxic cells include gamma/delta chain and CD4+ NK 1.1+ cells);and 2) increased antibody production.

Other embodiments of the present invention are generally directed toproviding an isolated antiviral peptide characterized by the amino acidsequence GDEPLENYLDTEYF (SEQ ID: NO 6)(-Gly-Asp-Glu-Pro-Leu-Glu-Asn-Tyr-Leu-Asp-Thr-Glu-Tyr-Phe-) (“IPF-6”)and a significant in vitro binding affinity for HIV-1 gp120, gp 41 andhuman CD4 cells. The peptide has anti-retroviral activity in vivo,particularly anti-HIV-1 activity. The peptide, referred to herein asIPF-6 (Inactivated Pepsinogen Fragment-6), was isolated from porcinepepsinogen, purified, and irreversibly inactivated for use in HIV-1prophylactic, therapeutic and diagnostic procedures. IPF-6 is expectedto have anti-retroviral activity in vivo, particularly inhibition ofHIV-1 entry into human CD4+ cells.

The exemplified peptide was obtained from porcine pepsinogen (FIG. 1) byisolation from a 45 kDa band of IPF preparation under gelelectrophoresis (FIG. 2). It may also be derived from pepsinogen fromany other source containing this sequence, or from any other peptides orproteins containing this sequence whereby suitable source pepsinogensare readily available commercially. Common laboratory methods andreagents for selectively cleaving intact proteins and for isolating andsequencing the cleaved peptides, such as the Erdman degradation process,may be used. The peptide may also be produced by peptide synthesis,using conventional methods. Moreover, genetically engineered constructsexpressing the sequence of interest are generally preferred, althoughchemical syntheses may also be used.

The peptides in the IPF fractions may be isolated and concentrated byany one of several techniques well-known to those skilled in the art,such as ammonium sulfate precipitation. The produced peptide isolate maybe purified by standard processes such as gel filtration and RP-HPLC,and inactivated by exposure to a neutral-to-alkaline environment ofabout pH 6.5 or greater or as otherwise known in the art. The peptidemay also be alkylated to increase immunogenicity if desired, forexample, by the process described for methylation in U.S. PatentApplication Publication US 2004/0018639 A 1. A HPLC chromatogram of thepurified, inactivated IPF-6 product in one embodiment of the inventionis shown in FIG. 3.

Homologues or analogues of the sequence which conserve at least criticalbinding site amino acid structures and functions and also conserve anydistal structural/functional residues essential for binding activity asdescribed herein may be substituted for the IPF of SEQ ID: NOS. 1-6(IPF-1-IPF-6. Variants of the sequences, including chemically modifiedderivatives, having a high sequence similarity will be generallypreferred, provided that binding activity is not significantly adverselyaffected. Residues superfluous to the disclosed function of the peptidesof the invention may be deleted or added with the same proviso. Modifiedsequences may be evaluated for conserved binding activity by, forexample, following the binding assays described herein or in theliterature. Numerous databanks are accessible for protein sequenceanalysis, such as those which combine sequence similarity with foldrecognition to predict functional equivalents. Binding properties(affinity, specificity, etc.) may also be evaluated by the bindingassays described below or other conventional assays, as known in theart.

BIACORE assays were used for binding affinity. See FIG. 6. Thistechnology measures mass concentration of biomolecules close to asurface. The surface is made specific by attaching one of theinteracting partners. Sample containing the other partner(s) flows overthe surface: when molecules from the sample bind to the interactantattached to the surface, the local concentration changes and an SPR(surface plasmon resonance) response is measured. The response isdirectly proportional to the mass of molecules that bind to the surface.The SPR response can be expressed by resonance units (RU). One RUrepresents change of 0.0001 in the angle of the intensity minimum whichis equivalent to a change in concentration of 1 pg/mm.

The exact conversion factor between RU and surface concentration dependson properties of the sensor surface and the nature of the moleculeresponsible for the concentration change. Assays tracking the binding ofIPF with CD4 cells, gp41, gp120, and human sera are very important todetect the formation of the super antigen responsible for the specificimmune response. That is, the high response of IPF fragments, measuredin Response Units, indicates a high utility as a specific binding agentfor components of HIV.

IPF-6 demonstrates binding in vitro with nonglycolysed fragment 579-601of the HIV-1 envelope protein gp41 subunit, with gp120 HIV-1 subunit,with human CD4+ cells and with human serum under gel electrophoresis(Biocor method) (see, FIGS. 4-8). The assays for these bindings wereconducted by The Institute for Molecular Medicine using standardprotocols. The spontaneous binding of IPF-6 with the gp41 subunit is aparticularly important biological property. Separately, under simpleagarose electrophoresis, IPF-6 and gp41 move in opposite directions.However, when they are mixed prior to electrophoresis, gp41 changesdirection and takes the direction of IPF-6. Quantitative analysis showedthat the binding capacity ratio of IPF-6 to gp41 was 1:0.66. That is,three molecules of IPF-6 bound two molecules of gp41 to form a complexwhich may function in vivo as, for example, a superantigen withsignificant anti-HIV-1 biological activity. Such antigen can be used asa bioassay reagent, in the production of mono- or polyclonal antibodies,in the manufacture of vaccines, and in other applications whereinantigens are conventionally employed.

While the mechanism of these binding events is not completelyunderstood, it is contemplated that exposure of HIV-1 to the IPF-6 ofthe present invention will effectively block gp41 and gp120 domainsessential for viral entry into CD4+ cells and inhibit viral infection,in vivo and in vitro. It is also contemplated that the IPF-6 of thepresent invention will effectively compete with HIV-1 for its CD4+ cellsurface binding sites and inhibit virus entry into these cells, in vivoand in vitro. Various in vitro protocols are known in the art forpredicting in vivo antiviral activity of compounds for inhibitingreplication of HIV, and these protocols may be used in connection withthe practice of the present invention. Exemplary art-recognized anti-HIVscreening assays are cited in U.S. Pat. No. 5,869,522, issued 9 Feb.1999 to Boyd et al., including those described in J. Virol. Methods,33:87-100, 1991; J. Natl. Cancer Inst., 81:577-586, 1992; J. Med. Chem.35:1978-1986, 1992, and Boyd, M. R., in AIDS Etiology: Diagnosis,Treatment, and Prevention, pp 305-319 (Lippincott, 1988, DeVita, V. T.,Jr., et al., eds).

In accordance with one aspect of the present invention, IPF-6 is used todiagnose viral infection, particularly HIV-1 infection. Bioassayssuitable for this purpose are well-known and routine. Typical of theseare assays based on competitive binding between, for example, a knownamount of a viral protein and a biological sample to be tested for thesame viral protein, using an excess of antiviral reagent capable ofspecifically binding with the known protein, such as an antibody. Amixture of these is incubated and the amount of bound complex calculatedand compared to that in a control mixture lacking the sample. Thepresence, if any, and amount of the viral protein in the sample can thenbe determined. There are numerous variations on this process, such assandwich assays, assays with immobilized reagent, assays using labeledreagent (e.g., ELISA, RIA, FIA), and so on. Whatever the variation,whether for detecting or quantifying complex, or for additionalreagents, or other modification, they all require a binding agent forthe unknown sample. Any of these routine binding assays for quantifyingor identifying an unknown sample may thus be used in the practice of thepresent invention by substituting IPF-6 as the antiviral binding agentfor samples to be tested for HIV-1 gp120, gp41, or infected CD4+T-cells.

In accordance with another aspect of the present invention, IPF-6 isused as a prophylactic or therapeutic to prevent or to treat HIVinfections. (Herein the term “HIV infections” refers to AIDS and ARC inaddition to viral infection per se unless otherwise noted). For in vivouse, the IPF compositions disclosed herein may be prepared foradministration by mixing it at the desired degree of purity with apharmaceutically-acceptable carrier suitable for the route ofadministration, as well-known in the art. Although the IPF compositionsdisclosed herein are desirably administered with an adjuvant in someapplications, in situations where a series of IPF doses areadministered, boosters with the respective IPF may not require adjuvant.Intramuscular or subcutaneous injections are presently the contemplatedroute for both therapeutic and prophylactic administration of the IPFcompositions disclosed herein. However, intravenous delivery, deliveryvia catheter or other surgical tubing, or other parenteral route mayalso be used. Alternative routes include oral routes for administeringtablets, liquid formulations and the like, as well as inhalation routes.Liquid formulations reconstituted from powder formulations may beutilized. The IPF compositions disclosed herein may also be administeredvia microspheres, liposomes, or other microparticulates, and viadelivery systems or sustained release formulations dispersed in certaintissues including blood.

The dosage administered of the IPF compositions as disclosed herein willdepend upon the properties of the formulation employed, e.g., itsbinding activity and in vivo plasma half-life, the concentration of IPFin the formulation, the administration route, the site and rate ofdosage, the clinical tolerance of the patient involved, the patient'scondition, and other considerations, as known in the art. Differentdosages may be utilized during a series of sequential treatments. Thepractitioner may administer an initial dose and then boost withrelatively smaller doses of IPF. The dosages of the IPF compositionsdisclosed herein may be combined with other HIV antivirals such as AZT.

EXAMPLES Isolation and Purification of Irreversibly-Inactive PepsinFraction

The following Examples are examples of methods for isolating, purifying,and characterizing the IPF compositions disclosed herein from active pigpepsinogen.

Example I Isolation and Inactivation of Pepsinogen Fragment

All the buffers and solutions used in this section were sterilized byfiltration. If needed, the buffers (0.2 N or 0.1 N HCl) were used toadjust the solutions. All the chemicals, including the distilled water,for the preparation of the buffers and solutions were USP Grade. Theratio of the pepsin to the buffers was 1:4 (weight/weight).

IPF was isolated from active pepsin (Sigma 1:10000) by ammonium sulfateprecipitation with centrifugation at about 4° C. The lyophilized pepsinpowder was dissolved in 0.14M sodium chloride (NaCl), 0.05M sodiumacetate (CH₃COONa. 3H₂0), 0.05M sodium citrate (C₆H₅O₇Na₂.2H₂O), and0.20N HCl (pH 2.8-3.2) buffer. The pH of the active pepsin suspensionwas then increased to 6.2-6.6 and the suspension was incubated for 30minutes. The suspension was then precipitated with a saturated solutionof (NH₄)₂SO₄. After degradation, the mixture was centrifuged (8000 RPMat 4° C.) for 60 minutes and the supernatant discarded. The pellet wasdissolved in a minimum quantity of 0.14M NaCl, and the resultingsolution was dialyzed for 18 hr against dialysis buffer: 0.1M NaCl, 0.1Msodium acetate, and 0.02M thimerozal USP, pH 6.8.

Example II Purification and Recovery of Irreversibly InactivatedPepsinogen Fragment

The purification of IPF included the following steps: dialysis,centrifugation, gel filtration, and reversed-phase HPLC.

After dialysis, the low molecular weight dialysate was centrifuged at15,000 rpm at 4□C for 60 minutes (Beckman rotor) with precipitation ofthe residual ammonium sulfate. The product was purified by gelfiltration to recover purified IPF from the crude mixture, and thenpurified by filtration on Bio-gel P10 or Sephadex G-75 gels (fromPharmacia Uppsala, Sweden), or 0.2μ SFCA membrane (Nalgene Labware,Rochester, N.Y.). Further purification was achieved by reversed phasehigh-performance liquid chromatography in an RP-HPLC system GOLD(Beckman) on C-18 columns (RP Ultrasphere 10 mm Spherical 80APreparative 21.2×150 mm) using gradient 30% acetonitrile diluted insterile water, HPLC-grade at 15% methanol HPLC-grade mobile phase.Detection 254 nm; flow rate 0.850 ml/min., solvent at pH 6.8. The finalpurification step included sterile filtration with Nalgen filters 0.45μ.The HPLC elution profile of the product showed one isolated peak, IPF(see FIG. 3).

Example III Determination of Molecular Weight

Molecular weight was determined by silver stained 13% non-reducingSDS-PAGE using the Laemmli method (Nature 227-680, 1970). The molecularweight standard demonstrated one peptide with a molecular weight of45.000 KD (FIG. 2). This band was isolated, and HPLC chromatogram (FIG.3) confirmed a single peptide in the band.

Example IV Assessment of Binding Activity

Samples of IPF (#18, 19, 20, and 21) were used to detect binding withgp120, gp41, CD4+ cells, and serum from a healthy patient. New chipswere coated with these proteins and Biacore assays for binding activitywere performed. These samples were diluted to 1:2000, 1:500 and 1:100.The results are shown in FIGS. 4, 5, 6, and 7. Sample #21 bound to alltarget proteins better than the other samples. The assay used a Biacore(Biacore AB, Uppsala, Sweden) system based on sensor chips which providesurface conditions for attaching molecules of interest, a microfluidicflow system for delivering samples to the surface, and a surface plasmaresponse (SPR) which detects mass concentration at the surface.SPR-based biosensors monitor interactions by measuring the mass ofmolecules bound to the surface. This response is expressed by resonanceunits (RU), whereby a change in concentration of 1 pg/mm is equivalentto a change of 0.0001 in the angle of intensity minimum, which equalsone RU. The exact conversion factor between RU depends upon theproperties of the sensor surface and the nature of the moleculeresponsible for the change in concentration. The assays demonstrate theformation of superantigen for provoking immune response.

Example V UV Absorption

Circular dichroism (CD) provides information about the secondarystructure of optically active materials. The far-UV or amide region(170-250 nm) is dominated by contributions of the peptide bonds, whereasCD bands in the near-UV region (250-300 nm) originate from the aromaticamino acids. The UV region of IPF was in the range of 252-260 nm.

Example VI Methylation of IPF

The purified IPF was methylated to form a methylated IPF preparation asfollows. IPF was mixed with CH2BrCOOH in sufficient quantity to producea final solution of 0.2 M CH2BrCOOH. For example, 2.78 g CH2BrCOOH wasdissolved in 10 ml distilled water and added to 100 ml IPF from the HPLCpurification step, containing 700 mg IPF (7 mg/ml). The pH of themixture was adjusted to and maintained at 7.2 with 0.1 M NaOH. Themixture was allowed to incubate from 6 to 8 hours. The IPF protein inthe resulting aqueous fragment was concentrated by ammonium sulfateprecipitation, using techniques known in the art. To 10 ml of themethylated IPF was added an equal volume of saturated ammonium sulfate.The mixture was refrigerated at 4 C for 12 hours and then centrifuged at15000 rpm for 60 minutes. The pellet was removed and dissolved in finalbuffer containing 0.1 M NaCl, 0.1 M sodium citrate, and 0.02Mthiodiglycol. The mixture was then dialyzed against the same buffer, toremove ammonium sulfate, for 24 hours.

Methylated IPF is lyophilized to form a slightly yellow powder. It isstable at room temperature but is preferably stored under refrigeration.The molecular weight of the IPF fragment was determined by silverstained 13% non-reducing SDS-PAGE (sodium dodecyl sulfate-polyacrylamidegel electrophoresis) using the Laemmli method (Laemmli, U. K., Nature,227:680 (1970)). Standards were: bovine serum albumin (66,000 molecularweight, MW); porcine heart fumarose (48,500 MW); bovine erythrocytescarbonic anhydrase (29,000 MW); bovine milk beta-lactoglobulin (18,400MW); and bovine milk α-lactalbumin (14,200 MW). The isoelectric point ofthe IPF composition is about 6.2 as determined by isoelectric focusing.Pepsin, in contrast, has a low pI.

Example VII Adjuvant

The IPF may be formulated in an aluminum hydroxide adjuvant. 1 ml of anIPF formulation contains: 4 mg IPF, 0.016 M AlP0.sub.4 (or 0.5 mgAl.sup.3+), 0.14 M NaCl, 0.004 M CH.sub.3COONa, 0.004 M KCl, pH 6.2.ALUM-aluminum hydroxide; Al(OH)₃. Aluminum hydroxide is a widely usedadjuvant, especially in commercial products such as vaccines. It is verywell suited for strong antigens. Many sources of aluminum hydroxides areavailable, e.g. Alhydrogel, Accurate Chemical & Scientific Co.,Westbury, N.Y.

Example VII Preparation of IPF Injection for Treating HIV

The inactivated pepsin fragment suspension may be prepared for injectinga preparation of highly purified inactivated pepsin fragment, such asthe IPF-6 fragment with a molecular weight of 45 KDa.

For example, the formulation may comprise (w/v) 0.4% inactivated pepsinfragment, 0.23% aluminum phosphate U.S.P., 1.29% sodium citrate U.S.P.,0.41% sodium acetate U.S.P. and water for injection to 100%. For a 1000ml batch: place 900 ml of U.S.P. sterile water into container,preferably glass. Add 12.9 g sodium citrate and mix until dissolved. Add4.1 sodium acetate and mix until dissolved. Add 4 g inactivated pepsinfragment, mix until a homogenous clear solution is obtained. Filter theresulting solution through a sterile 02. μm filter into a steriledepyrogenated 2 liter container with a sterile magnetic stirrer. Sterilefilter 55 ml of 0.016 M trisodium phosphate solution into the above 2liter sterile container. Sterile filter 50 ml of 0.016 M aluminumchloride solution into the 2 liter container, with the aluminum chloridebeing dispensed at a steady, drop by drop rate. Stir the resultinginactivated pepsin fragment suspension for 30 minutes at roomtemperature. Continue stirring for another 6 hours at 4° C. The sterileinactivated pepsin fragment suspension is ready to be filled intosterile 3 ml borosilicate vials.

The final 1 ml of the final IPF formulation may contain: 4 mg IPF(purity preferably >96%±0.290); 2.26 mg 0.016M AlPO₄ (or 0.5 mg Al⁺³);4.1 mg 0.004M CH₃COONa (sodium acetate); and 12.9 mg C₆H_(S)O₇ (sodiumcitrate); pH 6.2. In yet a further embodiment, the formulation maycomprise per vial, about 8 mg IPF, 4:52 mg aluminum phosphate, 1.0 mgaluminum, 25.8 mg sodium citrate and 8.2 mg sodium acetate. In oneregimen, 2 ml of this formulation makes up one vial with the dosage perpatient per day being 16 vials. During the regimen, the patient shouldbe monitored to assess the effectiveness of the regimen. CD+4 cellcounts are useful and common methodology for evaluating HIV infection,as are assays for antibody or T-cell titers.

Example VII IPF Formulation and Administration

The following is an example of a contemplated IPF formulation, dosageand administration schedule, which may be used with the IPF compositionsdisclosed herein:

The patient is administered an intramuscular injection containing 8 mgof the IPF composition (preferably 2 ml of a formulation containing 6mg/ml of IPF in a pharmaceutically acceptable solution) or 57 μg of IPFprotein per kg body weight of the patient. Each treatment courseconsists of 16 injections, with two injections on consecutive days perweek for 8 weeks. Three months after the last injection, if thepatient's condition warrants, the treatment regimen is repeated. Thetreatment regimen may be repeated until satisfactory results areobtained, e.g., a halt or delay in the progress of the infection ordisease, an alleviation of the infection or disease, or a cure isobtained. Preferably, in this application, IPF will be formulated withan aluminum hydroxide adjuvant.

Example VIV

Table 1 includes data from a preliminary study of a 54 year-old malepatient with 4^(th) stage pancreatic carcinoma treated with two cyclesof treatment with IPF, which were administered to the patient inSeptember, 2008. Unenhanced images of the liver were obtained at 5 mminterval and thickness. Following bolus of IV infusion of 125 ml ofnonionic contrast (Isovue 370) parameters used were: abdomen and pelvis5 mm thick helical scans; axial reconstructions were obtained at 2.5 mmslice thickness and 2 mm slice interval; patient received oral contast.Prior examination was on Apr. 18, 2008. In this study, the fulltreatment comprises three cycles of 16 vials of IPF each cycle.

In the second examination, tumor measurement was obtained with a CT scandone in November 2008. CT scan of the abdomen with limited imagingthrough the lower lungs showed that the patient had two very small focaldensities in the right middle lobe likle atelectasis. No pleuraleffusion were seen. In the liver is significant for multiple hypodesnselesions consistent with metastases. Many of these appear to decreaseslightly in size when compared to the prior examination. The lesion inthe posterior right lobe currently measures 35×22 mm. Previously itmeasured 43×33 mm. A lesion in the anterior left lobe currently measures18×17 mm. previously it measured 26×25. The other lesions all appearedto decrease in size. There was a hypodense lesion seen in the spleenslightly decreased consistent with a metastatic lesion. The pancreaticmass was not well demonstrated due to adjacent stomach. It measuredapproximately 40×48 mm slightly decreased in size. The patient has had aprior cholecystectomy. Left adrenal masses were demonstrated suggestingmetastases not significantly changed. The kidneys showed normalexcretion of contrast bilaterally. A large amount of ascites is presentnew compared with prior examination.

No definite retroperitoneal lymphadenopathy or pelvic lympadenopathy wasdemonstrated. Two small focal right middle lobe lung densities werelikely atelectasis. Multiple hypodense lesions consistent withmetastases all were slightly decreased when compared with the priorexamination. Splenic lesion had decreased. Left adrenal masses notsignificantly changed. Large amount of ascites present new. Pancreaticmass poorly demonstrated bu likely slightly decreased. The finding onthe CT scan showed that most of the lesion in the liver, pancreas andspleen had decreased in size. The patient's CA 19-9 at 19, returned tonormal.

The IPF composition used in this study comprised IPF-IL2 adjuvant,specifically the study was done using SEQ ID: NOS. 1 and 2. The RocheModular E170 CA 19-9 electrochemiluminescent immunoassay was used.

TABLE 1 Cancer Alkaline Antigen 19-9, Phosphatase, U/mL (ref range: U/L(ref Date 0-37 U/mL) range: 38-126) Jan. 15, 2008 31 101 Mar. 07, 200852 143 Mar. 27, 2008 116 * Apr. 24, 2008 97 235 May 07, 2008 52 * Jun.03, 2008 108 133 Jul. 15, 2008 68 141 Aug. 05, 2008 55 207 Oct. 07, 200822  95 Nov. 12, 2008 19 103

A person skilled in the art would appreciate that exemplary embodimentsdescribed hereinabove are merely illustrative of the general principlesof the present invention. Other modifications or variations may beemployed that are within the scope of the invention. Thus, by way ofexample, but not of limitation, alternative configurations may beutilized in accordance with the teachings herein. Accordingly, thedrawings and description are illustrative and not meant to be alimitation thereof.—

Moreover, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Thus, it is intended that the invention cover allembodiments and variations thereof as long as such embodiments andvariations come within the scope of the appended claims and theirequivalents.

1. An isolated irreversibly inactivated anti-cancer pepsin characterizedby the amino acid sequence of SEQ ID: NO. 1
 2. A composition comprisingthe isolated irreversibly inactivated anti-cancer pepsin of claim 1,further comprising a carri
 3. A composition for inferring immunityagainst malignant human cells, said composition comprising thecomposition of claim 2 combined with IL2
 4. A therapeutic compositioncomprising a complex of the composition of claim 1 bound to human gp96.5. A therapeutic composition comprising a complex of the composition ofclaim 1 bound to a receptor disposed on human gp96.
 6. The compositionof claim 5, where in the receptor is CD91.
 7. A 45 kD pepsinogenfragment consisting of the isolated irreversibly inactivated anti-cancerpepsin of claim 1, wherein said pepsinogen fragment has a bindingaffinity for human gp96.
 8. A composition comprising the therapeuticcomposition of claim 4, said composition further comprising IL2.
 9. Anisolated irreversibly inactivated anti-cancer pepsin characterized bythe amino acid sequence of SEQ ID: NO.
 2. 10. A composition comprisingthe isolated irreversibly inactivated anti-cancer pepsin of claim 9,further comprising a carrier.
 11. A therapeutic composition comprising acomplex of the composition of claim 9 bound to human gp96.
 12. Acomposition comprising the therapeutic composition of claim 11, saidcomposition further comprising IL2.
 13. A thirty one amino acidpepsinogen fragment having a binding affinity for human gp96.
 14. Anisolated irreversibly inactivated anti-cancer pepsin characterized bythe amino acid sequence of SEQ ID: NO.
 3. 15. A composition comprisingthe isolated irreversibly inactivated anti-cancer pepsin of claim 14,further comprising a carrier.
 16. A therapeutic composition comprising acomplex of the composition of claim 14 bound to human gp96.
 17. Thetherapeutic composition of claim 16, wherein the irreversiblyinactivated anti-cancer pepsin of claim has a molecular weight ofapproximately 13.5 kD.
 18. The composition of claim 16 furthercomprising IL2.
 19. A composition comprising an IPF gp96 complex,wherein said composition demonstrates superantigen activity.
 20. Acomplex of gp96 and IPF, wherein the complex activates majorhistocompatibility complex (HMC)-restricted manner.
 21. An isolatedirreversibly inactivated anti-cancer pepsin characterized by the aminoacid sequence of SEQ ID: NO.
 4. 22. A composition comprising theisolated irreversibly inactivated anti-cancer pepsin of claim 21,further comprising a carrier.
 23. A therapeutic composition comprising acomplex of the composition of claim 21 bound to human gp96.
 24. Anisolated irreversibly inactivated anti-cancer pepsin characterized bythe amino acid sequence of SEQ ID: NO.
 5. 25. A composition comprisingthe isolated irreversibly inactivated anti-cancer pepsin of claim 24,further comprising a carrier.
 26. A therapeutic composition comprising acomplex of the composition of claim 24 bound to human gp96.
 27. Thecomposition of claim 26, further comprising IL2.
 28. A method oftreating cancer, said method comprising the step of administering to apatient the therapeutic composition of claim
 4. 29. A method of treatingcancer, said method comprising the step of administering to a patientthe therapeutic composition of claim
 11. 30. A method of treatingcancer, said method comprising the step of administering to a patientthe therapeutic composition of claim
 16. 31. A method of treatingcancer, said method comprising the step of administering to a patientthe therapeutic composition of claim
 23. 32. A method of treatingcancer, said method comprising the step of administering to a patientthe therapeutic composition of claim 26.